Some biological traits are mediated by combinations of genes or proteins whose interactions are so complicated that we can not predict an organism's phenotype even with an intimate knowledge of its genotype. The twelve penicillin binding proteins (PBPs) of Escherichia coli form a model system for the study of such "complex phenotypes." The PBPs synthesize, modify and maintain the rigid peptidoglycan layer of the bacterial cell wall and are the targets of our most important single class of antibiotics, the beta-lactams. Nonetheless, despite decades of work, we do not know the detailed biological functions of these enzymes nor can we describe the biochemical pathways by which they operate. This information is becoming increasingly important with the rise of antibiotic resistant organisms. Our long term objective is to explain the structure, synthesis, and function of bacterial peptidoglycan so that more rational antimicrobial strategies can be devised. Therefore, we constructed 192 E. coli strains from which were deleted every possible combination of eight different PBPs. This comprehensive set of mutants allowed us to show that such a combinatorial genetic strategy produces results impossible to classic genetic approaches. Preliminary screening of the mutants revealed unusual and unanticipated phenotypes, including: capsule production, morphological aberrations, phage resistance, resistance to antibiotic- induced lysis, and temperature sensitivity. In most cases, the traits did not appear in cells with fewer than three or four mutations, and these phenotypes depended in a complex way on the combinations of active PBPs. We propose to complete the screening of this set of mutants for traits likely to be affected by a alterations in the peptidoglycan-e.g., in antibiotic-or chemically-induced autolysis, phage resistance, protein secretion, and in the morphogenesis of extracellular structures. In addition, analytical techniques will be adapted so that phenotypic predictions can be made from knowledge of the genotype in complex situations. Three significant results can be anticipated. First, we will identify new phenotypes in basic cellular processes in which the PBPs and peptidoglycan play fundamental biological roles. Second, we will understand better how the PBPs maintain the bacterial cell wall and how beta-lactam antibiotics induce its destruction. And third, the compilation of extensive and defined datasets will allow us to develop appropriate tools to investigate complex relations between genotype and phenotype.